Cellular Phenomena Research Articles

Abstract 279: Iatrogenic immunosuppression emplaces tacrolimus to drive squamous skin cancer progression and influence the microenvironment

Abstract One in thirteen organ transplant recipients (OTRs) develop post-transplant cutaneous squamous cell carcinoma (cSCC), leading to significant morbidity upon disease progression. Although chronic ultraviolet (UV) exposure primarily underlies keratinocyte carcinogenesis, long-term immunosuppression is a critical etiologic risk factor due to reduced systemic surveillance of dysplasia. Calcineurin inhibitors such as tacrolimus are commonly administered to suppress the immune system by blocking T-cell proliferation and preventing donor organ rejection. Contrarily, studies have evinced the off-target effects of immunosuppressives, namely azathioprine and cyclosporine, but not tacrolimus — the most widely used in all immunosuppression. In addition, the preclinical research literature has overlooked the contribution of stromal fibroblasts in the cSCC tumor microenvironment (TME). Hence, we hypothesize that tacrolimus exerts direct carcinogenic effects on keratinocytes and fibroblasts in the skin. We further postulate that tacrolimus enables a tumor-permissive microenvironment by activating dermal fibroblasts to cause aggressive and invasive keratinocyte carcinoma in OTRs. To examine this cellular crosstalk phenomenon, we used human keratinocytes, primary dermal fibroblasts and cSCC cells in mono- and co-cultures to mimic cutaneous and tumor complexities. Next, we assessed the molecular mechanisms utilized by tacrolimus to potentiate tumorigenesis pertinent to cell survival, proliferation and invasion. Herein, we show that tacrolimus promotes cell survival dose-dependently in cSCC cells. We also found that a fibroblast-cSCC spheroid co-culture significantly enhances cell invasion. In a wound-healing migration assay, tacrolimus increased the migratory capacity of normal keratinocytes and cSCC cells. However, cell proliferation remains nebulous due to cyclical dose-response effects in vitro. We then queried whether cell proliferation is affected in vivo using immunodeficient mice, and similar results were observed. We confirmed via immunoblotting that tacrolimus induced elevated expression of proliferation markers at distinct drug concentrations. Altogether, our findings illuminate keratinocyte-stroma interactions and particularly isolate the contributory role of tacrolimus in non-immune mediated cSCC pathogenesis. This knowledge suggests newer immunosuppressives that do not inhibit calcineurin activity may help relieve cutaneous malignancies in transplant recipients. Citation Format: Edwin Kumah, Yash Chhabra, Vania Wang, Agrani Dixit, Ashani Weeraratna, Kristin Bibee. Iatrogenic immunosuppression emplaces tacrolimus to drive squamous skin cancer progression and influence the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 279.

Cellular senescence promotes meibomian gland dysfunction in a chronic graft-versus-host disease mouse model

PurposeAging is a well-established risk factor for meibomian gland dysfunction (MGD). We previously reported an accelerated cellular senescence phenomenon in the lacrimal glands of a murine model of chronic graft-versus-host disease (cGVHD). Herein, we aimed to elucidate the relationship between cellular senescence and MGD in cGVHD mice, utilizing the senolytic agent ABT-263. MethodsA cGVHD mouse model was established through allogeneic bone marrow transplantation (BMT) from B10.D2 to BALB/c mice. Subsequently, cGVHD mice were treated with either ABT-263 or vehicle. The eyelids of recipients were analyzed at 4-week intervals post-BMT in both groups. ResultsMeibomian gland (MG) area was significantly smaller in cGVHD mice than in syngeneic control mice. ABT-263-treated mice retained a significantly larger MG area than their vehicle-treated counterparts. Pathological and immunohistochemical examinations revealed significant reductions in eyelid tissue inflammation and pathological fibrosis in the ABT-263 group compared to that in the vehicle-treated group. Additionally, expression of DNA damage markers, senescent cell markers, and senescence-associated secretory phenotype (SASP) factors was elevated in the eyelids of cGVHD mice compared with that in syngeneic mice. The expression of these cellular senescence-associated molecules was considerably suppressed in ABT-263-treated eyelids compared to that in vehicle-treated ones. ConclusionsCellular senescence, along with expression of SASP factors, exhibited increased activity in the eyelids, particularly in the MGs of cGVHD mice. ABT-263 mitigated the severity of MGD. These findings highlight the potential of targeting cellular senescence as an effective approach for MGD treatment in cGVHD.

Lipid droplets as substrates for protein phase separation

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

Saracatinib prompts hemin-induced K562 erythroid differentiation but suppresses erythropoiesis of hematopoietic stem cells.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs).We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified.These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

Phase separation-mediated biomolecular condensates and their relationship to tumor.

Phase separation is a cellular phenomenon where macromolecules aggregate or segregate, giving rise to biomolecular condensates resembling "droplets" and forming distinct, membrane-free compartments. This process is pervasive in biological cells, contributing to various essential cellular functions. However, when phase separation goes awry, leading to abnormal molecular aggregation, it can become a driving factor in the development of diseases, including tumor. Recent investigations have unveiled the intricate connection between dysregulated phase separation and tumor pathogenesis, highlighting its potential as a novel therapeutic target. This article provides an overview of recent phase separation research, with a particular emphasis on its role in tumor, its therapeutic implications, and outlines avenues for further exploration in this intriguing field.

Tumor-immune metaphenotypes orchestrate an evolutionary bottleneck that promotes metabolic transformation.

Metabolism plays a complex role in the evolution of cancerous tumors, including inducing a multifaceted effect on the immune system to aid immune escape. Immune escape is, by definition, a collective phenomenon by requiring the presence of two cell types interacting in close proximity: tumor and immune. The microenvironmental context of these interactions is influenced by the dynamic process of blood vessel growth and remodelling, creating heterogeneous patches of well-vascularized tumor or acidic niches. Here, we present a multiscale mathematical model that captures the phenotypic, vascular, microenvironmental, and spatial heterogeneity which shapes acid-mediated invasion and immune escape over a biologically-realistic time scale. The model explores several immune escape mechanisms such as i) acid inactivation of immune cells, ii) competition for glucose, and iii) inhibitory immune checkpoint receptor expression (PD-L1). We also explore the efficacy of anti-PD-L1 and sodium bicarbonate buffer agents for treatment. To aid in understanding immune escape as a collective cellular phenomenon, we define immune escape in the context of six collective phenotypes (termed "meta-phenotypes"): Self-Acidify, Mooch Acid, PD-L1 Attack, Mooch PD-L1, Proliferate Fast, and Starve Glucose. Fomenting a stronger immune response leads to initial benefits (additional cytotoxicity), but this advantage is offset by increased cell turnover that leads to accelerated evolution and the emergence of aggressive phenotypes. This creates a bimodal therapy landscape: either the immune system should be maximized for complete cure, or kept in check to avoid rapid evolution of invasive cells. These constraints are dependent on heterogeneity in vascular context, microenvironmental acidification, and the strength of immune response. This model helps to untangle the key constraints on evolutionary costs and benefits of three key phenotypic axes on tumor invasion and treatment: acid-resistance, glycolysis, and PD-L1 expression. The benefits of concomitant anti-PD-L1 and buffer treatments is a promising treatment strategy to limit the adverse effects of immune escape.

Post-polio syndrome is not a dysimmune condition.

Poliomyelitis is a global disabling disease affecting 12-20 million of people. Post poliomyelitis syndrome (PPS) may affect up to 80% of polio survivors: increased muscle weakness, pain, fatigue, functional decline. It relies on aging of an impaired neuro-muscular system with ongoing denervation processes. A late involvement of humoral or cellular pro-inflammatory phenomena is also suspected. To assess the dysimmune hypothesis of PPS by comparing lymphocyte subpopulations and humoral immune factors between PPS patients and controls. Cross-sectional study. Montpellier University Hospital. Forty-seven PPS and 27 healthy controls. PPS patients and controls were compared on their lymphocyte subpopulations and humoral immune factors (IL-1β, IL-6, IL-8, IL-17, IL-21, IL-22, IL-23, IFN-γ, TNF-α, GM-CSF, RANTES, MCP1, MIP-3a, IL-10, TGF-β, IL4, IL13). Patients were further compared according to their dominant clinical symptoms. Sample size guaranteed a power >90% for all comparisons. PPS patients and controls were comparable in gender, age and corpulence. Most patients had lower limb motor sequelae (N.=45, 95.7%), a minority had upper limb motor impairment (N.=16, 34.0%). Forty-five were able to walk (94%), 35/45 with technical aids. The median of the two-minute walking test was 110 meters (interquartile range 55; 132). Eighteen (38%) required help in their daily life. Their quality of life was low (SF36). All described an increased muscular weakness, 40 (85%) a general fatigue, and 39 (83%) muscular or joint pain. Blood count, serum electrolytes, T and B lymphocyte subpopulations and cytokines were comparable between patients and controls, except for creatine phospho kinase that was significantly higher in PPS patients. None of these variables differed between the 20/47 patients whose late main symptoms were pain or fatigue, and other patients. Our results suggest that PPS is not a dysimmune disease. Our results do not sustain immunotherapy for PPS. Our work suggest that PPS may be mostly linked to physiological age-related phenomena in a disabled neuromuscular condition. Thus, our results emphasize the role of prevention and elimination of aggravating factors to avoid late functional worsening, and the importance of rehabilitation programs that should be adapted to patients' specific conditions.

Marketable 1,3-dimethylamylamine and caffeine-based thermogenic supplements: Regulatory genotoxicity assessment through in vitro and in silico approaches

ABSTRACT The consumption of dietary supplements to enhance physical performance has increased significantly in the last century, especially thermogenic pre-workout supplements. Nevertheless, this industry has faced criticism for inadequate safety measures surveillance in regulatory issues regarding their products. The aims of our study were to investigate two pre-workout supplements with respect to (1) mutagenicity utilizing Salmonella/microsome assay; (2) genotoxicity employing cytokinesis-block micronucleus (CBMN) assay protocols; and (3) hepatocytoxicity using WST cell proliferation, activities of lactate dehydrogenase (LDH) and alkaline phosphatase using human liver carcinoma (HepG2) and mouse fibroblast (F C3H) cells. Oxidative stress was determined through glutathione (GSH) measurement and in silico for predictions of pharmacokinetics and toxicity for the most abundant isolated substances present in these supplements. Both supplements induced mutagenicity in all examined bacterial strains, especially in the presence of exogenous metabolism. Further, tested supplements significantly elevated the formation of micronuclei (MN) as well as other cellular phenomena. Concentration- and time-dependent curves were observed for hepatotoxicity in both studied cell lines. In addition, both supplements decreased levels of intracellular and extracellular GSH. In silico predictions showed that the isolated individual compounds failed to induce the observed outcomes. Our findings provide contributions to the molecular mechanisms underlying two pre-workout supplement-induced toxicity and the need for surveillance.

Abstract LB_C07: Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor-2 over-expression.  TNBC comprises 10-15% of all breast cancer cases and compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence and the chemotherapy agent doxorubicin (dox) remains a mainstay of treatment.  Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells can secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. Senescence is one potential mechanism of resistance to doxorubicin in breast cancer. Avoiding senescence-mediated resistance and promoting apoptosis is one strategy to improve response to dox in TNBC. Venetoclax is an orally bioavailable FDA approved BCL-2 inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate venetoclax in combination with dox to overcome senescence-mediated resistance and induce apoptosis. Experimental Procedures: TNBC cell lines were exposed to a no drug, dox, venetoclax or combination at increasing doses for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+.  Apoptosis at 48 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and with single agents.  In vitro cell survival following drug treatment for 24 hours was assessed via clonogenic assay.  In vitro senescence-associated β-gal was performed after 6 days drug treatment.  Athymic nude mice were subcutaneously injected with 5 million MDA-MB-231 cells for in vivo studies.  Mice were treated with either no drug, 60 mg/kg venetoclax PO QD, 1.5 mg/kg dox IP QW, or the combination. Results: The combination of venetoclax and dox resulted in synergy based on CellTiter-Glo data, which was used to calculate Bliss scores using Synergy Finder+ in CAL-51, MDA-MB-231, and CAL-120 TNBC cell lines. The dox and venetoclax combination caused a statistically significant increase in apoptosis measured by Annexin V staining when compared to single agents and control in all cell lines.  The combination resulted in a decrease in cellular survival in all cell lines tested as measured by the clonogenic assay.  Senescence-associated beta-galactosidase showed an increase in senescent cells for both dox and venetoclax treated cells, which was eradicated in combination. Dox and venetoclax were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to doxorubicin (p = 0.0031) and venetoclax alone (p = 0.0038) after 48 days of treatment. Conclusion: The addition of venetoclax to doxorubicin resulted in decreased cellular proliferation, increased apoptosis, and decreased senescence.  As a result, venetoclax is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Citation Format: Evan D Dus, Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Stacey M Bagby, Adrian T A Dominguez, Cameron A Binns, Todd M Pitts, Jennifer R Diamond. Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C07.

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis.

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles. This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (<1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers. When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

The life of the cell membrane: A paradigmatic reading from Deleuze and Guattari

While the Fluid Mosaic model (FMM) is widely accepted as an account of the cell membrane's structure-function, its inability to explain certain phenomena has led to the lipid rafts hypothesis (nanodomains) that spontaneous spatiotemporal enriched zones of sphingolipids-cholesterol-protein exist within the membrane. In this text, we propose a novel approach that conceives the cell membrane as a living entity. The questions regarding the FMM revolve around the fact that, although these molecular components are present in many cell types, the membrane does not react in the same way to every external agent; for example, a virus evokes a particular response: why is there some marked specificity of virus (or toxin) attack on one (or some) of these cell types and not to other cell types that nevertheless have a similar membrane protein constitution? The crucial question, to explain this selectivity, would be what determines the specificity of attack on some cells and not others? While FMN assumes a dynamism between macrostates at the intramolecular, intermolecular, and/or collective levels in the membrane, the approach of the lipid raft model presupposes a much greater and more complex dynamics of microstates (even nano-states) of these molecular components. In other words, it implies higher and instantaneous mobility as assemblages ("intentional") and thus, of the membrane itself (as a collective), in response to changes in the internal and external physicochemical environment over a broad spatiotemporal scale. This suggests a mechanism of membrane adaptation in the face of evolutionary constraints. In this text, we propose a paradigmatic approach, from Deleuze-Guattari's philosophy: to conceive the cell membrane as living and not as a mere molecular conglomerate with particular functions and mechanical processes between molecules. For this, we employ the functional concepts of territory and machinic assemblage, whence the vitality of the membrane would allow us to postulate instantaneous updates, within wider spatiotemporal scales in its composition in contrast with the model that dominates as a more plausible explanation nowadays, that does not include smaller spatiotemporal events. If we resort to the concept of territory and its different media components, we could offer a more plausible explanation of the vigorous dynamism in the composition of the cell membrane since it would allow more subtle and complex differentiations between media and thus make visible the constant and instant changes. We propose that the model of nanodomains, understood as a process of dynamic territorialization, offers a more complex and subtle explanation of the instantaneous changes in the cell membrane's composition. This approach expands the explanatory framework for cellular phenomena and reveals their spatiotemporal complexity in accordance with other research.

Development and Evaluation of Curcumin-loaded Niosomal Gel for Wound Healing in Rats

Background: The process of wound healing is a complicated series of organized biochemical and cellular phenomena that restore the integrity of the skin and subcutaneous tissue. A drug molecule can be transported to the site of action through a variety of carriers, including niosomes or non-ionic surfactant vesicles. They can trap medications that are both hydrophilic and hydrophobic. It has been demonstrated that curcumin has strong analgesic, anti-inflammatory, and wound-healing qualities. Methods: In the current study, an effort has been made to create a curcumin-loaded niosomal gel for efficient wound healing therapy. Utilizing non-ionic surfactants (Span 60, Span 20, Tween 20, and Tween 60) and cholesterol at various concentrations, a modified thin-film hydration process was used to create niosomal gels containing curcumin. Particle size, shape, entrapment effectiveness, deformability, and in vitro skin penetration were used to describe the vesicles. The 1% carbopol 940 gel was then made using the improved formulation. In wound-induced rats, the effectiveness of the in vivo wound healing was assessed. Results: The manufactured niosomes were discovered to be homogeneous in size and spherical in shape. According to the IR spectrum study, the medication and formulation additives did not interact. It was discovered that the niosomal gel (S-3) loaded with curcumin was not irritating to the skin. According to studies on in vivo wound healing, curcumin-loaded niosomal gel exhibited the highest percentage of wound contraction. Following 21 days of niosomal gel treatment, the histopathological evaluation found a noticeable improvement in the skin's histological architecture. Curcumin-loaded niosomal gel was easily made using the thin film hydration process. Conclusion: The results of this study demonstrate the potential of niosomal gel as a cutting-edge therapeutic strategy for wound healing.

Single-Cell Network-Based Drug Repositioning for Discovery of Therapies against Anti-Tumour Necrosis Factor-Resistant Crohn's Disease.

Primary and secondary non-response affects approximately 50% of patients with Crohn's disease treated with anti-tumour necrosis factor (TNF) monoclonal antibodies. To date, very little single cell research exists regarding drug repurposing in Crohn's disease. We aimed to elucidate the cellular phenomena underlying resistance to anti-TNF therapy in patients with Crohn's disease and to identify potential drug candidates for these patients. Single-cell transcriptome analyses were performed using data (GSE134809) from the Gene Expression Omnibus and Library of Integrated Network-Based Cellular Signatures L1000 Project. Data aligned to the Genome Reference Consortium Human Build 38 reference genome using the Cell Ranger software were processed using the Seurat package. To capture significant functional terms, gene ontology functional enrichment analysis was performed on the marker genes. For biological analysis, 93,893 cells were retained (median 20,163 genes). Through marker genes, seven major cell lineages were identified: B-cells, T-cells, natural killer cells, monocytes, endothelial cells, epithelial cells, and tissue stem cells. In the anti-TNF-resistant samples, the top 10 differentially expressed genes were HLA-DQB-1, IGHG1, RPS23, RPL7A, ARID5B, LTB, STAT1, NAMPT, COTL1, ISG20, IGHA1, IGKC, and JCHAIN, which were robustly distributed in all cell lineages, mainly in B-cells. Through molecular function analyses, we found that the biological functions of both monocyte and T-cell groups mainly involved immune-mediated functions. According to multi-cluster drug repurposing prediction, vorinostat is the top drug candidate for patients with anti-TNF-refractory Crohn's disease. Differences in cell populations and immune-related activity within tissues may influence the responsiveness of Crohn's disease to anti-TNF agents. Vorinostat may serve as a promising novel therapy for anti-TNF-resistant Crohn's disease.

Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

Molecular Mechanisms of Resistance to Ionizing Radiation in S. cerevisiae and Its Relationship with Aging, Oxidative Stress, and Antioxidant Activity.

The repair of the damage produced to the genome and proteome by the action of ionizing radiation, oxidizing agents, and during aging is important to maintain cellular homeostasis. Many of the metabolic pathways influence multiple processes. In this way, this work aims to study the relationship between resistance/response to ionizing radiation, cellular aging, and the response mechanisms to oxidative stress, free radicals, reactive oxygen species (ROS), and antioxidant activity in the yeast S. cerevisiae. Systems biology allows us to use tools that reveal the molecular mechanisms common to different cellular response phenomena. The results found indicate that homologous recombination, non-homologous end joining, and base excision repair pathways are the most important common processes necessary to maintain cellular homeostasis. The metabolic routes of longevity regulation are those that jointly contribute to the three phenomena studied. This study proposes eleven common biomarkers for response/resistance to ionizing radiation and aging (EXO1, MEC1, MRE11, RAD27, RAD50, RAD51, RAD52, RAD55, RAD9, SGS1, YKU70) and two biomarkers for response/resistance to radiation and oxidative stress, free radicals, ROS, and antioxidant activity (NTG1, OGG1). In addition, it is important to highlight that the HSP104 protein could be a good biomarker common to the three phenomena studied.

Fluid dynamic design for mitigating undesired cell effects and its application to testis cell response testing to endocrine disruptors

Microfluidic devices have emerged as powerful tools for cell-based experiments, offering a controlled microenvironment that mimic the conditions within the body. Numerous cell experiment studies have successfully utilized microfluidic channels to achieve various new scientific discoveries. However, it has been often overlooked that undesired and unnoticed propagation of cellular molecules in such bio-microfluidic channel systems can have a negative impact on the experimental results. Thus, more careful designing is required to minimize such unwanted issues through deeper understanding and careful control of chemically and physically predominant factors at the microscopic scale. In this paper, we introduce a new approach to improve microfluidic channel design, specifically targeting the mitigation of the aforementioned challenges. To minimize the occurrence of undesired cell positioning upstream from the main test section where a concentration gradient field locates, an additional narrow port structure was devised between the microfluidic upstream channel and each inlet reservoir. This port also functioned as a passive lock that hold the flow at rest via fluid-air surface tension, which facilitated manual movement of the device even when cell attachment was not achieved completely. To demonstrate the practicability of the system, we conducted experiments and diffusion simulations on the effect of endocrine disruptors on germ cells. To this end, a bisphenol-A (BPA) concentration gradient was generated in the main channel of the system at BPA concentrations ranging from 120.8 μM to 79.3 μM, and the proliferation of GC-1 cells in the BPA gradient environment was quantitatively evaluated. The features and concepts of the introduced design is to minimize unexpected and ignored error sources, which will be one of the issues to be considered in the development of microfluidic systems to explore extremely delicate cellular phenomena.

The GTPase Rab21 is required for neuronal development and migration in the cerebral cortex.

Development of the mammalian neocortex requires proper inside-out migration of developing cortical neurons from the germinal ventricular zone toward the cortical plate. The mechanics of this migration requires precise coordination of different cellular phenomena including cytoskeleton dynamics, membrane trafficking, and cell adhesion. The small GTPases play a central role in all these events. The small GTPase Rab21 regulates migration and neurite growth in developing neurons. Moreover, regulators and effectors of Rab21 have been implicated in brain pathologies with cortical malformations, suggesting a key function for the Rab21 signaling pathway in cortical development. Mechanistically, it has been posited that Rab21 influences cell migration by controlling the trafficking of endocytic vesicles containing adhesion molecules. However, direct evidence of the participation of Rab21 or its mechanism of action in the regulation of cortical migration is still incomplete. In this study, we demonstrate that Rab21 plays a critical role in the differentiation and migration of pyramidal neurons by regulating the levels of the amyloid precursor protein on the neuronal cell surface. Rab21 loss of function increased the levels of membrane-exposed APP, resulting in impaired cortical neuronal differentiation and migration. These findings further our understanding of the processes governing the development of the cerebral cortex and shed light onto the molecular mechanisms behind cortical development disorders derived from the malfunctioning of Rab21 signaling effectors.

Immunosenescence as a convergence pathway in neurodegeneration.

Immunity refers to the body's defense mechanism to protect itself against illness or to produce antibodies against pathogens. Senescence is a cellular phenomenon that integrates a sustainable growth restriction, other phenotypic abnormalities and including a pro-inflammatory secretome. It is highly involved in regulating developmental stages, tissue homeostasis, and tumor proliferation monitoring. Contemporary experimental reports imply that abolition of senescent cells employing evolved genetic and therapeutic approaches augment the chances of survival and boosts the health span of an individual. Immunosenescence is considered as a process in which dysfunction of the immune system occurs with aging and greatly includes remodeling of lymphoid organs. This in turn causes fluctuations in the immune function of the elderly that has strict relation with the expansion of autoimmune diseases, infections, malignant tumors and neurodegenerative disorders. The interaction of the nervous and immune systems during aging is marked by bi-directional influence and mutual correlation of variations. The enhanced systemic inflammatory condition in the elderly, and the neuronal immune cell activity can be modulated by inflamm-aging and peripheral immunosenescence resulting in chronic low-grade inflammatory processes in the central Nervous system known as neuro-inflammaging. For example, glia excitation by cytokines and glia pro-inflammatory productions contribute significantly to memory injury as well as in acute systemic inflammation, which is associated with high levels of Tumor necrosis factor -α and a rise in cognitive decline. In recent years its role in the pathology of Alzheimer's disease has caught research interest to a large extent. This article reviews the connection concerning the immune and nervous systems and highlights how immunosenescence and inflamm-aging can affect neurodegenerative disorders.

Nuclear proteostasis imbalance in laminopathy-associated premature aging diseases.

Laminopathies are a group of rare genetic disorders with heterogeneous clinical phenotypes such as premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, and so on. The cellular phenomena associated with laminopathy invariably show disruption of nucleoskeleton of lamina due to deregulated expression, localization, function, and interaction of mutant lamin proteins. Impaired spatial and temporal tethering of lamin proteins to the lamina or nucleoplasmic aggregation of lamins are the primary molecular events that can trigger nuclear proteotoxicity by modulating differential protein-protein interactions, sequestering quality control proteins, and initiating a cascade of abnormal post-translational modifications. Clearly, laminopathic cells exhibit moderate to high nuclear proteotoxicity, raising the question of whether an imbalance in nuclear proteostasis is involved in laminopathic diseases, particularly in diseases of early aging such as HGPS and laminopathy-associated premature aging. Here, we review nuclear proteostasis and its deregulation in the context of lamin proteins and laminopathies.

Convolutional Neural Network for Segmenting Micro-X-ray Computed Tomography Images of Wood Cellular Structures

To further enhance the performance of wood products, improved tools are needed to study in situ cellular scale phenomena like mechanical deformations and moisture swelling. Micro-X-ray computed tomography (μXCT) using brilliant synchrotron light sources now has the spatial and temporal resolution for real-time visualization of phenomena in three-dimensional cellular structures. However, the tradeoff for speed includes the loss of intensity contrast between different types of materials within the imaged structure, such as cell wall and air in wood. This loss of contrast prevents traditional histogram-based segmentation methods from being used effectively. A new convolutional neural network (CNN) approach was therefore developed to segment fast μXCT images of wood into cell wall and air volumes. The fast μXCT and segmentation were demonstrated in the study of moisture swelling in loblolly pine (Pinus taeda) earlywood and latewood cellular structures conditioned at 0%, 33%, 75%, and 95% relative humidity (RH). The CNN segmentation results had a mean intersection over union (IoU) metric accuracy of 96%. Initial analysis of the swelling in the latewood revealed cell walls swelled about 25% when conditioned from 0% to 95% RH. Additionally, the widths of ray cell lumina in the transverse plane of latewood could be observed to increase at higher RH. The segmentation method presented here will facilitate future quantitative analyses in in situ μXCT studies of wood and other similar cellular materials.